The highly specific binding nature of immunoglobulins, e.g., antibodies and antibody-like molecules (e.g., camelid immunoglobulin or immunoglobulin new antigen receptors (IgNARs) from cartilaginous fish) or proteins comprising antigen binding domains thereof makes them particularly suitable for delivering molecules to specific targets in a subject. For example, immunoglobulins or proteins comprising antigen binding domains thereof can be conjugated to cytotoxic or cytostatic compounds e.g., drugs, to kill or inhibit growth of cells, such as tumour cells (Lambert, 2005). Such a conjugate facilitates targeted delivery of the cytotoxic or cytostatic compounds to cells expressing the antigen to which the immunoglobulin or fragment binds, rather than non-specifically throughout a subject. Such conjugates can permit use of compounds that are generally toxic to a subject by ensuring the delivery of toxic levels of the compound to the site at which it is required rather than systemically within a subject. Furthermore, conjugation of antibodies or proteins comprising antigen binding domains thereof to detectable compounds, such as fluorophores or radioisotopes facilitates detection of target molecules within a subject, for example to facilitate detection of diseased cells such as cancer cells, e.g., using in vivo, imaging-based methods.
Conventional means of linking a compound to an antibody or a protein comprising antigen binding domain generally leads to a heterogeneous mixture of molecules where the compounds are attached at a number of sites on the antibody. For example, compounds have typically been conjugated to an antibody or protein comprising antigen binding domains thereof through the often-numerous lysine residues in the antibody or antigen binding domain, generating a heterogeneous antibody-compound conjugate mixture. Depending on reaction conditions used, the heterogeneous mixture typically contains a distribution of conjugates with from 0 to about 8, or more, attached compounds. In addition, within each subgroup of conjugates with a particular integer ratio of compounds to antibody or protein there is a potentially heterogeneous mixture where the compound is attached at various sites on the antibody or protein. Analytical and preparative methods are inadequate to separate and characterize the various conjugate species within the heterogeneous mixture resulting from a conjugation reaction.
Furthermore, non-specific conjugation of a compound to an antibody or protein comprising an antigen binding domain thereof may reduce or completely prevent binding of the antibody/protein to an antigen, for example, if the compound is conjugated to a region required for antigen binding. This risk is increased in proteins that comprise antigen binding domains that are far smaller than an intact antibody in which there may be few residues suitable for conjugation that are not important for antigen binding. For example, proteins comprising little more than antigen binding domains of an antibody have few sites to which a compound can be conjugated without reducing or preventing antigen binding.
Carbohydrate(s) on the Fc region of an antibody is a natural site for attaching compounds. Generally, the carbohydrate is modified by periodate oxidation to generate reactive aldehydes, which can then be used to attach reactive amine containing compounds by Schiff base formation. As the aldehydes can react with amine groups, reactions are carried out at low pH so that lysine residues in the antibody or antigen binding domain are protonated and unreactive. Hydrazide groups are most suitable for attachment to the aldehydes generated since they are reactive at low pH to form a hydrazone linkage. The linkage can then be further stabilized by reduction with sodium cyanoborohydride to form a hydrazine linkage (Rodwell et al, 1986). Disadvantages of this approach include the harsh conditions required for linkage which can damage and aggregate some antibody molecules. For example, methionine residues present in some antibody variable regions may be particularly susceptible to oxidation by periodate which can lead to loss of antigen binding avidity. Histidine and/or tryptophan residues are also susceptible to oxidation. Furthermore, many proteins comprising antigen binding domains of an antibody do not necessarily comprise a Fc region, meaning that they cannot be conjugated to a compound using the foregoing process.
Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997). Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent attachments to ligands or to form new intramolecular disulfide bonds. However, inserting or substituting cysteine thiol groups into a protein is potentially problematic, particularly in the case of those which are relatively accessible for reaction or oxidation, i.e., positioned at sites useful for conjugation of a compound. This is because, in concentrated solutions of the protein, whether in the periplasm of Escherichia coli, culture supernatants, or partially or completely purified protein, cysteine residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and hence protein aggregates. Such protein aggregation often leads to poor yields of isolated protein that is in a useful form, e.g., having a desired biological activity. Furthermore, the protein oxidatively can form an intramolecular disulfide bond between the newly engineered cysteine and an existing cysteine residue, which can render the protein inactive or non-specific by misfolding or loss of tertiary structure. Each of the foregoing problems are exacerbated in antibodies and proteins comprising antigen binding domains thereof which generally comprise several cysteine residues that bond with one another to ensure correct folding and stability and, as a consequence antigen binding activity.
It will be clear to the skilled artisan from the foregoing that there is a need in the art for proteins comprising antigen binding domains of immunoglobulins that are modified so as to permit simple conjugation of a compound thereto. Preferred proteins will facilitate recombinant production in a variety of systems, preferably without resulting in considerably levels of multimeric aggregates linked by intermolecular bonds.